Pulmonary embolism is the commonest preventable cause of death in hospitalized patients. Early detection of its most common precursor, venous thrombosis of the lower extremities, would permit prompt anticoagulant therapy and reduce the frequency of embolism. However, the potential for successful treatment of established pulmonary embolism is limited by the short time between onset of symptoms and death in the majority of patients who die of massive pulmonary embolism. Secondly, most patients with massive pulmonary embolism do not have preceding clinical signs of minor venous thromboembolism, even though postmortem examination shows that most of them do have associated leg vein thrombosis. Unfortunately, clinial diagnosis of venous thrombosis and phlebographic technique are neither specific nor reliable during acute phase of thrombophlebitis. There is an urgent need for a simple, rapid and reliable means of detecting venous thrombosis. A number of techniques for screening large numbers of high-risk patients are being evaluated at present. The most promising appears to be radioiodinated fibrinogen labeled with .sup.125 I. Because of low energy gamma photon and long physical half life, the use of .sup.125 I-fibrinogen is limited to surface monitoring technique. It is not a scintillation imaging agent. Other limitations include high percentage of false positive results due to its inability to distinguish between superficial and deep vein thrombi, and its sensitivity to fibrin in hematoma and inflammatory exudate. Autologous human fibrinogen labeled with .sup.131 I has been advocated recently as a thrombi scanning agent.
Another important group of plasma protein which may have significant clinical applications in Nuclear Medicine is immunoglobulins or antibodies. The immunoglobulins are protein molecules that carry antibody activity against specific antigens. With the possible exception of natural antibody, antibodies arise in response to foreign substances such as microorganisms, toxin or other foreign matter introduced into the body. The immunoglobulins comprise a heterogeneous group of proteins, chiefly gamma or beta globulins, which account for approximately 20% of the total plasma proteins. The presence of malignant tumors can also cause the production of antibodies within the host in response to the insult. Thus, radiolabeled autologous immunoglobulin isolated from patient's own serum which contains the specific antibody offers the best and specific means of detecting infectious foci or tumors. Early detection of these leisons is extremely important in reducing the high morbidity and mortality rate. The use of .sup.131 I-labeled antigen or antibody for tumor imaging in man have been reported in the literature.
Various methods of labeling plasma proteins with .sup.125 I or .sup.131 I have been published in recent years. The most commonly used chemical method is radio-iodination of the protein in the presence of chloramine-T or iodine monochloride. The labeling yield, however, is low and varies from 50-70%. In order to be clinically useful, the desired radiolabeled protein must undergo a long and tedious separation and purification process. The radionuclide .sup.131 I has other disadvantages. Among these are: emission of high energy beta and gamma photons which is not compatible with existing commercial display means; a long physical half life of 8 days; excessive irradiation to the patients; and finally, the dosage of any .sup.131 I-labeled compounds must be given in very minute microcurie(uCi) quantity.
Compounds labeled with .sup.99m Tc which eliminate most of the undesirable properties of the radioiodinated radiopharmaceuticals have been found extremely useful in biological studies and medical diagnosis. The radionuclide, .sup.99m Tc-technetium, has many advantages. It is a pure gamma emitter with a relative short half life of 6 hours. The gamma photon of 140 KeV energy is compatible with existing conventional scintillation imaging equipments. .sup.99m Tc-labeled radiopharmaceuticals can be safely administered to the patients with a much larger dose than radioiodinated compounds but produces a minimal amount of radiation health hazard. Human proteins such as serum albumin labeled with .sup.99m Tc has been used clinically in placenta localization, cardiac scan and cisternography. More recently, there is increasing scientific and medical interest in .sup.99m Tc-labeled human fibrinogen and antibody for the localization and detection of thrombi, infectious foci and tumors. Unfortunately, a more wide spread use of these radioactive tracer materials has been restricted because; (1) a simple and reliable chemical method of labeling protein with .sup.99m Tc at physiological condition which preserves the physiobiological properties of the protein has not been developed; (2) current .sup. 99m Tc-labeling technology using acid reduction of the radionuclide in the presence of a reducing agent causes complete denaturation of the proteins; (3) the labeling yield is low with many radioactive impurities as well as free or unbound .sup.99m Tc: (4) the possibility of hepatitis transmission and antigenic reactions.
Recent literature mention labeling serum albumin with .sup.99m Tc by a chemical process in the presence of a reducing agent such as stannous chloride (SnCl.sub.2.2H.sub.2 O) or stannous tartrate (see U.S. Pat. No. 3,725,295 to Eckelman et al and U.S. Pat. No. 4,042,676 to Molenski et al), but the results have never been very satisfactory. According to the labeling methodology, .sup.99m Tc(+7) in the stable form of sodium pertechnetate(Na.sup.99m TcO.sub.4) is first reduced to a chemically active +4 or +5 valence state with a reducing agent in 0.5-1 N HCl at a pH of less than 2. A diluted solution of the albumin is added to the reduced .sup.99m Tc/SnCl.sub.2 acidic mixture with subsequent binding of the radionuclide to the protein ligand. The final mixture is than readjusted to pH 6-7 with a suitable buffer. The labeling mechanism is not known. Since the optimal condition of preserving the physiobiological properties of the protein is at a very narrow pH range of 7-7.4, proteins labeled by the above described chemical method is completely denatured.
The enzymes streptokinase and urokinase which are proteins, have been labeled with .sup.99m Tc using similar technique. (see U.S. Pat. No. 3,812,245 to Dugan and Dugan, MA, Kozzar, JJ, et al, J. Nucl. Med. 14 233, 1973) The labeling yields of these proteins are extremely low. Purification of these radioactive proteins requires a tedious process of removing large amount of free or unbound .sup.99m Tc, insoluble tin particles in the form of .sup.99m Tc-stannous hydroxide(.sup.99m Tc-Sn(OH).sub.4), and other protein degradation products. (see Duffy MJ and Duffy GJ, J. Nucl. Med. 18: 483, 1977 and Person, BRR and Kempe, V. J. Nucl. Med. 16: 474, 1975) .sup.99m Tc-streptokinase and .sup.99m Tc-urokinase have claimed to be effective in localizing preformed clots of the deep veins. However, they are ineffective in documenting early stage of acute thrombophlebitis. Both enzymes are antigenic in man.
An alternate approach for labeling proteins by chemical means has been patented in 1978 but has never been reported in any scientific scientific literature (see U.S. Pat. No. 4,057,617 to Abramovici et al). According to this invention, the proteins antibody and fibrinogen are labeled with .sup.99m Tc at pH 11.6. A careful analysis of the labeling methodology reveals many flaws. Stannous chloride dissolved in dilute hydrochloric acid(HCl) or acetic acid is known to be a powerful reducing agent for the reduction of .sup.99m TcO.sub.4.sup.-. Increasing the pH from 2 to 11.6 will not cause further reduction of .sup.99m Tc. On the contrary, during the process of pH adjustment, insoluble radioactive collodial particles, stannous hydroxide, will form when alkali such as 0.1 N NaOH is added to a solution containing SnCl.sub.2 and reduced .sup.99m Tc. The problems encountered by labeling proteins at alkaline pH condition is similar to the acidic chemical method, namely; protein denaturation, formation of insoluble stannous particles, protein degradation byproduct, free or unbound .sup.99m Tc and low yield.
Significant protein denaturation occurs with earlier electrolytic method of labeling serum albumin and fibrinogen with .sup.99m Tc using zirconium electrodes in acid medium (see U.S. Pat. No. 3,784,453 to Dworkin et al; Benjamin, PP, Int. J. Appl. Rad. Isotopes 20: 187, 1969; Dworkin, HJ and Gutkowski, RF, J. Nucl. Med. 12: 562, 1971 and Wong, DW and Mishkin, F, J. Nucl. Med. 16: 347, 1975). The labeling methodology requires the addition of the protein to be labeled to an acidic medium (pH 1.8) during electrolysis which leads to subsequent decomposition of the labeled product. Recently, an improved electrolytic method of labeling plasma proteins has been developed (see Wong, DW, J. Labeled Comp. Radiopharmaceuticals 14: 603, 1978 and Wong, DW and Huang, TT, Int. J. Appl. Rad. Isotopes 28:719, 1977). These proteins are labeled at physiological conditions, thus avoiding harsh treatment of the protein molecules and preserving the physiobiological properties. The labeling mechanism is not well understood. The tagging of .sup.99m Tc to pure protein appears to involve a chemically active .sup.99m Tc-(Zr)citrate complex species with high protein binding capacity. The latter is formed following initial reduction of .sup.99m TcO.sub.4.sup.- by Zr.sup.++ ions as a result of electrolysis and by the addition of trisodium citrate/NaOH buffer during pH adjustment. In the presence of a pure protein, such as fibrinogen or immunoglobulin, .sup.99m Tc quickly binds to the protein ligand. Whether the entire complex binds to the protein ligand or acts only as a transferring agent for reduced .sup.99m Tc for the final labeling has not been determined. Further investigation of the improved electrolytic technique indicates that similar complex species can be prepared by chemical means with stannous chloride or stannous tartrate under similar conditions. The resultant .sup.99m Tc-(Sn)citrate complex species is effective in tagging plasma proteins with superior labeling efficiency and reproducibility. The labeling mechanism of the chemical method has not been determined. It is assumed that protein binding involves the reaction of the .sup.99m Tc-(Sn)-citrate complex species with the protein ligand similar to the .sup.99m Tc-(Zr)-citrate reaction (see Wong, DW, Mishkin, F and Lee, T, Int. J. Appl. Rad. Isotopes 29: 251, 1978).